This invention generally relates to the field of new dyes, dye compositions, and methods of detection of nucleic acids in red blood cells by flow cytometry by employing fluorescent, nucleic acid binding dyes. More particularly, this invention provides a reagent and method for enumeration of reticulocytes and nucleated red blood cells.
Analysis of red blood cells and enumeration of its immature sub populations are a valuable components of diagnostic hematology. For example, enumeration of reticulocytes, i.e., the immature erythrocytes, in human peripheral blood is useful in the diagnoses of hemorrhage, anemia, monitoring bone marrow transplantation and for monitoring patients undergoing chemotherapy and other disorders involving blood cell production [U.S. Pat. No. 5,360,739; H. Shapiro, Practical Flow Cytometry, 3rd edit., 1995; Wiley-Liss, New York; Davis et al, (1990) Pathobiol., 58:99-106; Hoy, (1990) Bailliere""s Clin. Haemat., 3:977-988; H. J. Tanke, Reticulocytes and Mature Erythrocytes in Flow Cytometry in Haematology (1992) Academic Press Ltd., pp. 75-93]. Because reticulocytes contain ribonucleic acid (RNA), if stained with RNA binding excitable dyes, these cells fluoresce when illuminated by a light source of appropriate wavelength. RNA binding dyes have been used to distinguish reticulocytes from mature red blood cells (RBCs) which lack RNA.
Distribution of fluorescence intensities of a relatively large reticulocyte population can be determined by flow cytometry in a fast and reliable manner, and different maturation stages of reticulocytes, as reflected by differences in RNA content, can be distinguished.
The use of red-excitable dyes is desirable because such dyes are detected by excitation with relatively inexpensive diode or HeNe lasers. However, initial efforts in the prior art to employ diode lasers and red-excited dyes for rapid flow cytometric analyses of reticulocytes were not successful. Yamamoto, U.S. Pat. No. 5,563,070 suggested that the addition of large quantities of TO-PRO-3, a red-excitable dye, followed by a 30 minute incubation, stained RNA inside living reticulocytes. Such a method, however, is not practical for routine analysis of reticulocytes in clinical laboratories that require high sample throughput because sample preparation time is long and cost per test is high due to the large amount of dye required to stain each sample.
A red-excitable dye called Thiazole Blue (TB) has been described in U.S. Pat. No. 4,957,870. However, as described in this patent (U.S. Pat. No. 4,957,870), this dye also requires long periods of incubation, of about 30 minutes.
Akai et al., U.S. Pat. No. 5,821,127 have also described the preparation of fluorescent dyes which are capable of detecting reticulocytes using inexpensive detectors via fluorescence in the red region. However the samples require incubation at elevated temperatures of about 40xc2x0 C.
More recently, U.S. Pat. No. 5,994,138, described staining reticulocytes via the use of a red-excitable dye in combination with a detergent and an ionophore at elevated temperatures of about 35xc2x0 C. However, staining reticulocytes was not successful when ambient temperatures were utilized.
Fan et. al (U.S. Pat. No. 5,411,891, U.S. Pat. No. 5,360,739) describes clearly that the specific binding constant between a dye and reticulocyte RNA and the rate of penetration of the dye are different for each dye and that it is impossible to predict under what conditions a particular dye may rapidly penetrate red cell membrane and stain reticulocytes. This was further supported by Akai et. al (U.S. Pat. No. 5,821,127).
Thus, there exists a need in the art for dyes, compositions and methods which enable rapid staining of intracellular RNA at room temperatures via the use of dyes that are excitable in the red region and can use inexpensive and readily available red-illumination instruments. In addition there exists a need in the art for dye compositions and methods that are applicable not only to red-excitable dyes, but also to dyes excitable at other wavelengths, such as in the blue wavelength. By doing so, ready and accurate detection of reticulocytes can be accomplished without particular restriction of the excitation wavelength.
In addition to the reticulocytes, another type of immature red blood cells that are important in clinical diagnostics are the nucleated red blood cells (NRBCs). In contrast to reticulocytes, NRBCs contain DNA. NRBCs normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, NRBCs also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBC. Since NRBCs contain DNA, fluorescence detection and enumeration of NRBCs can be possible by staining these cells with dyes that recognize DNA. Thus, there exists a need in the art for dyes, and compositions which enable staining of both intracellular DNA and RNA, so that both NRBCs and reticulocytes can be enumerated via the use of the same dye employing different methods.
In one aspect, the present invention provides three groups of novel dyes that are set forth as Group I, Group II, and Group III with:
Group I having the general formula of: 
wherein n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 is CH2(CH2)mOH,
wherein m is 0, 1, 2, or 3; X is O, S, or C(CH3)2; R is CH3, CH(CH3)2, CH2CH2OH, alkyl, alkylsulfonate, or hydroxyalkyl; and Bxe2x88x92 is a counteranion.
Group II having the general formula of: 
wherein, n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 is CH2(CH2)mOAc, wherein m is 0, 1, 2, or 3; X is O, S, or C(CH3)2; R is CH3, CH(CH3)2, CH2CH2OAc, alkyl, alkylsulfonate, or hydroxyalkyl and Bxe2x88x92 is a counteranion;
and,
Group-III having the following general formula: 
wherein n is 0, 1, or 2; R1 is H, alkyl, or an alkoxy group; R2 is CH2(CH2)mOH, or CH3 and wherein m is 0, 1, 2, or 3; X is O, S, or C(CH3)2Bxe2x88x92 is a counter anion, and R3, R4, R5, R6 are various substituents as represented by the formulae for the specific compounds Dye-3 through Dye-6.
In another aspect, the invention provides a reagent containing a dye of the invention and a solvent.
In yet another aspect, the invention provides compositions and methods for facilitating rapid transport of dye molecules through a cell membrane. Such rapid staining requires that a sample be contacted with a dye composition of the invention in the presence of at least one surfactant and optionally, a sulfonic acid or a salt thereof.
Other aspects and advantages of this invention will be readily apparent from the detailed description of the invention.